3D High Throughput Model to Predict Drug Efficacy in Fibrosis Progression vs Reversal

摘要：3D高通量模型预测肝纤维化进展与逆转的药物疗效 特发性肺纤维化(IPF)是最无情的进展性和致命性纤维性肺疾病，它 对老年人的影响不成比例。尽管最近有两种药物获得了FDA对IPF的批准，但这些 药物只能适度减缓肺部衰退的进程，并不能改善患者的生活质量。 目前还没有可用的治疗方法可以“逆转”纤维化。尽管许多团体努力开发 在IPF治疗方面，进展非常缓慢。该提案侧重于以下两个可能的原因 这些困难：(1)目前的临床前筛查模型不能可靠地预测药物的成功 (2)尽管IPF被广泛认为是一种与年龄相关的疾病，但药物治疗 没有针对与年龄相关的病理机制。 现有的病理性纤维化是一个“纤维增生性”的过程，并没有导致有效的间质性纤维化。 治疗。这项建议结合了成纤维细胞老化方面的专业知识和正在开发的IPF治疗新药 (Hecker Lab)拥有用于微型生物打印和3D细胞分析的尖端技术(Takayama Lab) 建立高通量表型细胞筛选试验，以确定纤维化逆转的有效性。这个 建议的研究将利用正常的“对照”、衰老的“衰老”和IPF的人肺成纤维细胞在小的 数字，以生物工程的高通量表型分析，将在21天内评估纤维化。 将使用双水相系统(ATPS)对这些细胞进行生物打印，以产生微小规模的收缩 体积比常规分析小几个数量级的分析。重要的是 该项目将在已经收缩的富含细胞的凝胶周围反复微型打印新鲜的胶原蛋白，以使 反复宫缩超过21天。所提出的模型将使第一个高通量表型 筛选试验能够确定候选药物对纤维化进展和 反转。新的细胞测试将被验证其识别纤维化逆转药物的能力 “Noxindoline”是一种高度选择性的NOX4抑制剂，目前正由Hecker实验室进行临床前开发。 Noxindoline是由Hecker实验室通过研究NOX4的年龄相关性变化而确定的 导致持续的氧化还原失衡，并促进衰老和抗凋亡 肌成纤维细胞。该提案假设目前的治疗方法(宁替达尼和吡非尼酮)将抑制 促纤维化表型的进展(但不能逆转)，而诺辛多林治疗将促进 已确立的促纤维化表型的逆转。目标是： 目的：利用非衰老细胞建立高通量的肝纤维化进展生物印迹细胞检测方法 目的：监测衰老细胞和IPF患者细胞的纤维化进程和逆转

ABSTRACT: 3D High Throughput Model to Predict Drug Efficacy in Fibrosis Progression vs Reversal Idiopathic pulmonary fibrosis (IPF) is the most relentlessly progressive and fatal fibrotic lung disorder, which disproportionately affects the elderly. Although two drugs have recently gained FDA-approval for IPF, these drugs only moderately slow the progression of lung decline and do not improve quality of life for patients. There are no available therapies that can `reverse' fibrosis. Despite efforts by numerous groups to develop IPF treatments, progress has been aggravatingly slow. This proposal focuses on two possible reasons for these difficulties: (1) Current pre-clinical screening models fail to reliably predict the success of drug candidates in humans, and (2) Although IPF is widely regarded as an age-related disease, drug treatments have not targeted age-associated pathologic mechanisms. The existing paradigm, that pathologic fibrosis is a “fibro-proliferative” process, has not led to effective IPF treatments. This proposal integrates expertise in fibroblast aging and novel IPF therapeutics in development (Hecker lab) with cutting edge technologies for microscale bioprinting and 3D cell assays (Takayama lab) to develop a high throughput phenotypic cellular screening assay to determine efficacy for fibrosis reversal. The proposed studies will utilize normal “control”, aged “senescent”, and IPF human lung fibroblasts in small numbers to bioengineer a high-throughput phenotypic assay that will evaluate fibrosis over a 21 day period. An aqueous two phase system (ATPS) bioprinting of these cells will be used to create microscale contraction assays that are several order of magnitude smaller in volume compared to conventional assays. Importantly, the project will repeatedly micro-print fresh collagen around already contracted cell-laden gels to enable repeated contractions over 21 days. The proposed model will enable the first high-throughput phenotypic screening assay with the capability to determine a drug candidate's efficacy for fibrosis progression and reversal. The new cellular assay will be validated for its ability to identify fibrosis reversal drugs using “Noxindoline” a highly selective Nox4 inhibitor that is currently in preclinical development by the Hecker lab. Noxindoline was identified by the Hecker lab through studies of age-dependent alterations in Nox4 that results in a sustained redox imbalance, and promotes senescence and apoptosis-resistance of myofibroblasts. The proposal hypothesizes that current therapies (Nintedanib and Pirfenidone) will inhibit the progression of pro-fibrotic phenotypes (but not reversal), whereas treatment with Noxindoline will promote the reversal of established pro-fibrotic phenotypes. The aims are: Aim1: Develop high throughput bioprinted cellular assay for fibrosis progression using non-senescent cells Aim2: Monitor fibrosis progression and reversal of senescent cells and IPF patient cells